Here we report that structural changes in gonadal basement membranes during sex differentiation in the frog Rana rugosa are revealed using an antibody to its laminin component. Immunohistochemical staining indicated that the first sexual dimorphism appeared in testicular cords and ovarian cavities in differentiating gonads of tadpoles at St. 25-3W, three weeks after they reached St. 25. During development, as the testis enlarged, testicular cord partitions appeared to form by invagination of the testicular epithelium. Ovarian cavities also increased in volume. Laminin-positive basement membranes initially surrounded a partial surface of oocytes close to the ovarian cavity, fully covering growing oocytes by St. X. Laminin-reactive signals were present in somatic cells outside seminiferous tubules in the testis and outside oocytes in one-year-old frogs. BrdU-labeling showed that the number of dividing germ cells increased continuously in male gonads but increased in females only up to St. V, declining at St. X and thereafter. The number of dividing germ cells declined when the basement membranes had fully covered the oocytes. Together, these findings suggest that the first sexual dimorphism in the gonad of R. rugosa first appears as a structural change in the basement membranes. Finally, we speculate that the basement membrane on the surface of oocytes may affect their proliferation in this species.

The Pat1 gene is expressed in the immature oocytes of Xenopus, and is reportedly involved in regulating the translation of maternal mRNAs required for oocyte-maturation. However, it is still unknown when Pat1a first appears in the differentiating ovary of amphibians. To address this issue, we isolated the full-length Pat1a cDNA from the frog Rana rugosa and examined its expression in the differentiating ovary of this frog. Among eight different tissues examined, the Pat1a mRNA was detectable in only the ovary. When frozen sections from the ovaries of tadpoles at various stages of development were immunostained for Vasa-a germ cell-specific protein-and Pat1a, Vasa-immunopositive signals were observed in all of the germ cells, whereas Pat1a signals were confined to the growing oocytes (50-200 μm in diameter), and absent from small germ cells (<50 μm in diameter). Forty days after testosterone injection into tadpoles to induce female-to-male sex-reversal, Pat1a-immunoreactive oocytes had disappeared completely from the sex-reversed gonad, but Vasa-positive small germ cells persisted. Thus, Pat1a would be a good marker for identifying the sexual status of the sex-reversing gonad in amphibians. In addition, fluorescence in situ hybridization analysis showed Pat1a to have an autosomal locus, suggesting that Pat1a transcription is probably regulated by a tissue-specific transcription factor in R. rugosa.

We cloned a cDNA encoding Vasa, a member of the DEAD (Asp-Glu-Ala-Asp) family of proteins, from the ovary of the frog Rana rugosa. Comparative alignment of amino acid sequences with known Vasa from several species of vertebrate showed that the R. rugosa orthologue shares eight conserved regions with Vasa from other vertebrates. Vasa gene expression was restricted to the testis and ovary among ten different tissues examined. Vasa expression showed no sexual dimorphism during sex determination in R. rugosa, but became higher in the ovary thereafter. By Western blot analysis, a single Vasa band with a molecular weight of 80.9 kDa was detected. The same antibody immunohistochemically detected Vasa in a few cells in the embryonic endoderm at stage 15; the beginning of closure of neural folds, and in the cytoplasm of spermatogonia in the testis, and oocytes in the ovary of tadpoles at stage XX; marked by one or both forelegs protruded. Together, these results suggest that Vasa is a highly specific marker of germ cells and hence useful for studies of germ cell specification and function in amphibians as it already is in other species of both invertebrates and vertebrates such as Drosophila and zebrafish.